Persistent inhibition of cell respiration by nitric oxide: Crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione

Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.

Loss of ovarian function promotes angiogenesis in human ovarian carcinoma

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.

Luteinizing hormone induction of ovarian tumors: Oligogenic differences between mouse strains dictates tumor disposition

The use of fertility drugs has continued to grow since their introduction in the 1960s. Accompanying this increase has been the speculation that repetitive use of these drugs can cause ovarian tumors or cancer. We recently reported that transgenic mice with chronically elevated luteinizing hormone (LH), an analog of which is commonly used in fertility regimens, develop granulosa cell (GC) tumors. In this report we show that LH induction of these tumors is highly dependent on genetic background. In CF-1 mice, chronically elevated LH invariably causes GC tumors by 5 months of age. However, in hybrid mice generated by crossing CF-1 males with C57BL/6, SJL, or CD-1 females, elevated levels of this same hormone cause a completely different phenotype resembling a luteoma of pregnancy. We also show that three genes likely control these alternative hormonal responses. This clinical correlate of elevated LH reveals remarkably distinct, strain-dependent, ovarian phenotypes. In addition, these results support the rare incidence of GC tumors in the human population, and suggest that the ability of certain fertility drugs to cause ovarian tumors may depend on an individual's genetic predisposition.

A serologically identified tumor antigen encoded by a homeobox gene promotes growth of ovarian epithelial cells

Ovarian carcinomas are thought to arise from cells of the ovarian surface epithelium by mechanisms that are poorly understood. Molecules associated with neoplasia are potentially immunogenic, but few ovarian tumor antigens have been identified. Because ovarian carcinomas can elicit humoral responses in patients, we searched for novel tumor antigens by immunoscreening a cDNA expression library with ovarian cancer patient serum. Seven clones corresponding to the homeobox gene HOXB7 were isolated. ELISAs using purified recombinant HOXB7 protein revealed significant serologic reactivity to HOXB7 in 13 of 39 ovarian cancer patients and in only one of 29 healthy women (P < 0.0001). Ovarian carcinomas were found to express HOXB7 at markedly higher levels than normal ovarian surface epithelium, suggesting that immunogenicity of HOXB7 in patients could be associated with its elevated expression in ovarian carcinomas. Overexpression of HOXB7 in immortalized normal ovarian surface epithelial cells dramatically enhanced cellular proliferation. Furthermore, HOXB7 overexpression increased intracellular accumulation and secretion of basic fibroblast growth factor, a potent angiogenic and mitogenic factor. These results reveal HOXB7 as a tumor antigen whose up-regulated expression could play a significant role in promoting growth and development of ovarian carcinomas.

Selective response of ternary complex factor Sap1a to different mitogen-activated protein kinase subgroups.

Mitogenic and stres signals results in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases. A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1, which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals. A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which is not clear. Here we show that Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs. Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs. Moreover, Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress. These results suggest that Sap1a and Elk-1 have distinct physiological functions.

Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch.

In studies of variants of the P(ant) promoter of bacteriophage P22, the Arc protein was found not only to slow the rate at which RNA polymerase forms open complexes but also to accelerate the rate at which the enzyme clears the promoter. These dual activities permit Arc, bound at a single operator subsite, to act as an activator or as a repressor of different promoter variants. For example, Arc activates a P(ant) variant for which promoter clearance is rate limiting in the presence and absence of Arc but represses a closely related variant for which open-complex formation becomes rate limiting in the presence of Arc. The acceleration of promoter clearance by Arc requires occupancy of the operator subsite proximal to the -35 region and is diminished when Arc bears a mutation in Arg-23, a residue that makes a DNA-backbone contact in the operator complex.

Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes.

Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage.

Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies.

The x-ray structure of the complex of a catalytic antibody Fab fragment with a phosphonate transition-state analog has been determined. The antibody (CNJ206) catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and substrate specificity. Comparison of this structure with that of the uncomplexed Fab fragment suggests hapten-induced conformational changes: the shape of the combining site changes from a shallow groove in the uncomplexed Fab to a deep pocket where the hapten is buried. Three hydrogen-bond donors appear to stabilize the charged phosphonate group of the hapten: two NH groups of the heavy (H) chain complementarity-determining region 3 (H3 CDR) polypeptide chain and the side-chain of histidine-H35 in the H chain (His-H35) in the H1 CDR. The combining site shows striking structural similarities to that of antibody 17E8, which also has esterase activity. Both catalytic antibody ("abzyme") structures suggest that oxyanion stabilization plays a significant role in their rate acceleration. Additional catalytic groups that improve efficiency are not necessarily induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten.

Yeast global transcriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme.

Sin4 and Rgr1 proteins, previously shown by genetic studies to play both positive and negative roles in the transcriptional regulation of many genes, are identified here as components of mediator and RNA polymerase II holoenzyme complexes. Results with Sin4 deletion and Rgr1 truncation strains indicate the association of these proteins in a subcomplex comprising Sin4, Rgr1, Gal11, and a 50-kDa polypeptide. Taken together with the previous genetic evidence, our findings point to a role of the mediator in repression as well as in transcriptional activation.

Tissue-engineered cells producing complex recombinant proteins inhibit ovarian cancer in vivo

Techniques of tissue engineering and cell and molecular biology were used to create a biodegradable scaffold for transfected cells to produce complex proteins. Mullerian Inhibiting Substance (MIS) causes regression of Mullerian ducts in the mammalian embryo. MIS also causes regression in vitro of ovarian tumor cell lines and primary cells from ovarian carcinomas, which derive from Mullerian structures. In a strategy to circumvent the complicated purification protocols for MIS, Chinese hamster ovary cells transfected with the human MIS gene were seeded onto biodegradable polymers of polyglycolic acid fibers and secretion of MIS confirmed. The polymer-cell graft was implanted into the right ovarian pedicle of severe combined immunodeficient mice. Serum MIS in the mice rose to supraphysiologic levels over time. One week after implantation of the polymer-cell graft, IGROV-1 human tumors were implanted under the renal capsule of the left kidney. Growth of the IGROV-1 tumors was significantly inhibited in the animals with a polymer-cell graft of MIS-producing cells, compared with controls. This novel MIS delivery system could have broader applications for other inhibitory agents not amenable to efficient purification and provides in vivo evidence for a role of MIS in the treatment of ovarian cancer.

Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex

The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament—a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex.

Regulation of T cell receptor (TCR) β gene expression by CD3 complex signaling in immature thymocytes: Implications for TCRβ allelic exclusion

During αβ thymocyte development, clonotype-independent CD3 complexes are expressed at the cell surface before the pre-T cell receptor (TCR). Signaling through clonotype-independent CD3 complexes is required for expression of rearranged TCRβ genes. On expression of a TCRβ polypeptide chain, the pre-TCR is assembled, and TCRβ locus allelic exclusion is established. We investigated the putative contribution of clonotype-independent CD3 complex signaling to TCRβ locus allelic exclusion in mice single-deficient or double-deficient for CD3ζ/η and/or p56lck. These mice display defects in the expression of endogenous TCRβ genes in immature thymocytes, proportional to the severity of CD3 complex malfunction. Exclusion of endogenous TCRβ VDJ (variable, diversity, joining) rearrangements by a functional TCRβ transgene was severely compromised in the single-deficient and double-deficient mutant mice. In contrast to wild-type mice, most of the CD25+ double-negative (DN) thymocytes of the mutant mice failed to express the TCRβ transgene, suggesting defective expression of the TCRβ transgene similar to endogenous TCRβ genes. In the mutant mice, a proportion of CD25+ DN thymocytes that failed to express the transgene expressed endogenous TCRβ polypeptide chains. Many double-positive cells of the mutant mice coexpressed endogenous and transgenic TCRβ chains or more than one endogenous TCRβ chain. The data suggest that signaling through clonotype-independent CD3 complexes may contribute to allelic exclusion of the TCRβ locus by inducing the expression of rearranged TCRβ genes in CD25+ DN thymocytes.

Characterization of human telomerase complex

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called “telomeres” to the growing ends of chromosomal DNA. Characterization of mammalian telomerase has been elusive because of its low level of expression. We describe a bioinformatics approach to enrich and characterize the human telomerase complex. Using local sequence homology search methods, we detected similarity of the Tetrahymena p80 subunit of telomerase with the autoantigen Ro60. Antibodies to Ro60 immunoprecipitated the telomerase activity. Ro60 and p80 proteins were cross-recognizable by antibodies to either protein. Telomerase activity and the RNA component of telomerase complex were localized to a doublet in a native gel from the Ro60 antibody-precipitated material. The enriched material showed specific binding to a TTA GGG probe in vitro in an RNA template-dependent manner. Polyclonal antibodies to the doublet also immunoprecipitated the telomerase activity. These results suggest an evolutionary conservation of the telomerase proteins.

Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1α and its dual block by ovarian steroids

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1α (IL-1α) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1α and repress MMP-1 production by IL-1α-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1α and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1α is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1α, thereby exerting an effective control via a “double-block” mechanism.